Lipid-containing structures in the form of micelles, liposomes and other visicles, iscoms (immune-stimulating complex/particles), iscom matrices, etc. have been reported as effective carriers of pharmacologically and/or immunologically active substances or molecule complexes. See for example WO-A1-90/03184 (Morein et. al., Clin. Immunother. Review, 1995; Kersten et. al., Iscom--Liposome Review, 1995). In many cases, immunization of laboratory animals with such lipid-containing structures, in which various antigens have been incorporated, have been shown to give rise to an increased immune response to the referred antigens as compared to the immune response obtained after immunization using a corresponding antigen in a free form.
Iscom and iscom matrices are documented as effective carriers of antigens and adjuvant molecules to enhance the immunogenicity of small and large molecules (antigens), i e to make them strongly immunogenic both when they are applied parenterally and locally, (topically) on mucous surfaces. The iscom has unique properties being effective after mucosal intranasal adminstration. It is well-documented (Morein et. al., Clin. Immunother. 3, 1995, 461-475) that both iscoms with incorporated antigens (usually protein) and iscoms as carriers, for example small antigens such as oligopeptides or as exemplified by biotin, effectively evoke immune response to these large or small molecules.
Iscom matrices (and iscom) have well-documented, built-in, adjuvant activity that potentiates antibody-mediated as well as cell-mediated immune responses to the co-administered antigens. Iscom evokes cell-mediated immune response under both Class I and Class II restriction.
Cholera is the most serious of all the diarrhea diseases and is caused by the Vibrio cholera bacteria in group 1. These bacteria colonize in the small intenstine of human beings and secrete an exotoxin protein known as the cholera toxin. This toxin binds to and is absorbed by cells in the mucous membranes and causes an intensive secretion of electrolytes and water from the cells, which leads to the grave cases of diarrhea, dehydration, and metabolic acidosis which characterize cholera.
Similar diseases can be caused by so-called "enterotoxic" (ET) cholibacteria, but the symptoms are usually milder. Such bacteria often cause diarrhea in young individuals among humans and practically all kinds of animals, including pigs and cattle. These diarrheas, which can give rise to great economic losses for the livestock industry, are caused partly by a heat-labile toxin (LT) similar to the cholera toxin (CT). These toxins are so similar that they bind to the same receptors.
The structures of CT and LT are well defined in regards to structure and function. They are oligomeric proteins consisting of one part that binds to the cholera toxin receptor, namely the B part, which in turn consists of five subunits which each have an approximate mole weight of 11,600 and form a pentamer ring. The A subunit is a proteolytic split polypeptide with a molecular weight of approximately 28,000, consisting of two disulfid-conjugated fragments. The larger A1 fragment contains toxin-enzyme activity, while the smaller A2 fragment joins the A1 fragment with the B5 ring. CT binds with high affinity to a class of receptors that exist on the surface of the so-called brush-border membranes in the small intestine, as well as to the plasma membrane of most mammalian cells. The GM1 gangliosid constitute the receptor for CT (Holmgren et. al., Infect. Immun. 38, 424-433). LT also binds to GM1.
CT and LT, respectively, are both important components in the subunit vaccines that are intended to evoke protection gains cholera and enterotoxic cholibacteria. In the case of intestinal infections, it is of special interest to evoke local protection exerted by, among other things, secretory IgA in the intestinal membrane. CT and LT are both considered well suited as targeting molecules in adjuvant formulations for vaccines intended for adminstration in the intestinal and respiratory tracts (Morein. Lovgren and Cox, 1966), with, among other things, having the capacity to induce a secretory IgA response that is an important component in the protection. The B subunit of CT and LT have attracted a good deal of interest as carrier molecules and even as universal vector systems for oral vaccines (Mucosal Handbook Immunology, eds Ogra, P. L., Lamm Me, Mc Ghee Jr., Mestechy J., Strober W. Bienenstock J., 1994). The interest has increased even more because it has been shown that the conjugate between CTB and other antigens not only give rise to immune response in the local intestinal mucosal membranes, but also to a limited extent in other remote mucous membranes, such as the salivary glands, the lungs, the genital tract, and in the blood (Handbook mucosal, 1994). The problem with CTB and LTB is that they have a low (inate)-capacity to potentiate their own strong, protective immune response against the cholera toxin or against LT, or against the antigen that they are modified to be a carrier for. They thus have a low adjuvant activity in relation to the immunomodulatory and immunopotentiating effect (Morein, Lovgren and Cox, 1966).
CTB and LTB are used experimentally as carriers of antigen with the purpose of evoking, through local application (orally), local immune response in the mucous membranes of the digestive tract as well as in other mucous membranes through gut-associated lymphatic traffic (GALT) or through direct application on other mucous membranes such as the respiratory tract. CTB and LTB have targeting capacity, which means that they are considered to steer and localize both themselves and the antigens they may carry to the lymphatic system in the intestinal tract, which means to M-cells in Payer's patches, to lamina propria (LP), and to the lymphatic system in the intestines and in other mucous membranes through GALT or through direct application on these mucous membranes, for example in the respiratory tract.
The following unsolved difficulties exist regarding using CTB and LTB for local immunization:
1. CTB and LTB have by their own relatively low immunogenicity and a low immunoenhancing capacity, requiring a need to be potentiated with an adjuvant component to obtain optimal effect. In other words, this involves both their own immunogenicity and their immunoenhancing effect to the antigens that they may have carried with them. Their value as adjuvants is limited to "targeting", while supplementary adjuvant activities in the form of immunomodulatory and immunoenhancing capacities are required in order to attain optimal immunogenicity.
2. There are limitations to conjugating antigens to CTB and LTB with particularly high physical or economic yield, since only a limited number of amino groups and/or carboxy groups can be activated without seriously reducing their values as antigen or as carriers in mucous membranes, and target themselves and the accompanying antigens to the lymphatic organs and cells to evoke immune response. Even if a sufficient number of coupling groups are available on a carrier molecule, it is well known that it is difficult to attain the desired economic yield from such constructions because of the insufficient yield. For example, often no more than 15-20% of the available antigens are coupled in reaction to the carrier molecule.
3. CTB and LTB have a limited space for chemically coupling of larger molecules, because they can block functional epitopes that are necessary for targeting the complexes to the lymphatic organs and cells.